• Authors: Wei C. et al.
  • Year: 2021
  • Journal: Neurosci Bull s12264-021-00804-7
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T


HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin, transfected with appropriate expression plasmids using jetOPTIMUS (Polyplus-transfection, France), and incubated in a humidified 5% CO2 atmosphere at 37°C for 48 h. The cells were harvested in phosphate-buffered saline (PBS), spun down, and re-suspended on ice with lysis buffer [50 mmol/L Tris/HCl (pH 8.0), 200 mmol/L NaCl, 1% Triton X-100, complete proteinase inhibitors (Roche), and EDTA]. The proteins were solubilized by rotating the cell lysates at 4°C for 20 min on a rotating wheel, and the supernatants were then cleared by centrifugation at 12,000 g for 15 min at 4°C. The supernatant lysate was incubated with 2 µg anti-HA or anti-FLAG co-injected beads overnight at 4°C. The beads were washed four times with the lysis buffer. The bound proteins were eluted with 2× Laemmli sample buffer (Sigma–Aldrich) and loaded onto sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS-PAGE) for immunoblotting using rabbit anti-HA (1:2000) and mouse anti-Flag (1:2000).


The radial migration of cortical pyramidal neurons (PNs) during corticogenesis is necessary for establishing a multilayered cerebral cortex. Neuronal migration defects are considered a critical etiology of neurodevelopmental disorders, including autism spectrum disorders (ASDs), schizophrenia, epilepsy, and intellectual disability (ID). TRIO is a high-risk candidate gene for ASDs and ID. However, its role in embryonic radial migration and the etiology of ASDs and ID are not fully understood. In this study, we found that the in vivo conditional knockout or in utero knockout of Trio in excitatory precursors in the neocortex caused aberrant polarity and halted the migration of late-born PNs. Further investigation of the underlying mechanism revealed that the interaction of the Trio N-terminal SH3 domain with Myosin X mediated the adherence of migrating neurons to radial glial fibers through regulating the membrane location of neuronal cadherin (N-cadherin). Also, independent or synergistic overexpression of RAC1 and RHOA showed different phenotypic recoveries of the abnormal neuronal migration by affecting the morphological transition and/or the glial fiber-dependent locomotion. Taken together, our findings clarify a novel mechanism of Trio in regulating N-cadherin cell surface expression via the interaction of Myosin X with its N-terminal SH3 domain. These results suggest the vital roles of the guanine nucleotide exchange factor 1 (GEF1) and GEF2 domains in regulating radial migration by activating their Rho GTPase effectors in both distinct and cooperative manners, which might be associated with the abnormal phenotypes in neurodevelopmental disorders.